The major long-term objectives of this research proposal are to characterize the normal mammary tissue stem cells (MaSC) in the mouse, and determine how, and/or whether, MaSC participate in cancer development. These results in the mouse will be compared to human breast tumor types for extrapolating results to the human system. A mouse transgenic model in which the enhanced green fluorescent protein (GFP) is expressed in several stem/progenitor cell populations of the embryo and adult. In the adult mammary tissue, two unique GFP+ stem cell types have been observed during puberty, coincident with mammary ductal outgrowth, and again in early to late pregnancy, prior to the generation of alveolar/lobular structures. The GFP+ cells in puberty correspond to the cap cells in the growing end of the terminal end buds, which form the initial mammary ductal network. In early pregnancy the GFP+ cells appear as alveolar buds, which direct formation of alveoli/lobules necessary for milk production. The prospective labeling with GFP offers a one-step isolation of the GFP+ cell populations. These GFP+ cells are MaSC as demonstrated by transplantation outgrowth, lineage tracing using Cre/LoxP technology, and self- renewal experiments. The research proposed in this application will focus on three specific Aims, which will first, characterize the MaSC niche based on expected niche properties, determine Notch-dependent regulation of MaSC fate, and identify MaSC functions by stem cell ablation experiments. Secondly, the prospective identification of MaSC allows the direct test of the cancer stem cell hypothesis. Experiments in the second Aim will determine whether Wnt1 and ErbB2 (also called neu or HER2) mammary tumors contain GFP+ tumor cells, which contain the primary tumor-initiating activity. In addition, the target cell, within all mammary tissue epithelial cells, will be identified by cell isolation and direct in vitro transformation. Tumor cell readout will follow transplantation into mammary fat pads for outgrowth. The third Aim, will tie together GFP+ murine mammary tumors with gene expression-based human mammary tumor classifications. MicroRNAs in these GFP+ murine mammary tumors will be identified for further analysis of signaling and potential therapeutic use. PUBLIC HEALTH RELEVANCE: The prospective identification and characterization of mammary stem cells has enabled experiments to be performed, which were not previously possible. Therefore, results from this project will generate new insights into mammary stem cells and how they may contribute to normal mammary tissue and to cancer development. Addressing the cancer stem cell hypothesis will clarify present uncertainties in the target cells for which therapies should be directed, while gene expression data will draw parallels between mouse and human mammary cancers. These studies represent a new and promising paradigm for breast cancer research.